This invention relates to specific binding assays in which two receptors or binding partners are employed. In such assays a first receptor is bound by a second receptor, with the first receptor being capable of reversibly binding either a ligand or, still further, another receptor to the exclusion of structurally related substances. For the purposes of this invention such assays will be termed double receptor specific binding assays.
Receptors are generally proteins, although other substances having reversible specific binding affinity for a ligand or for a labelled analogue thereof are equally useful. The most commonly used receptors are antibodies because they can be raised to bind any desired ligand. However, other binding proteins are included within the meaning of receptor, e.g., intrinsic factor, thyroxine binding globulin, cortisol binding protein, folate binding protein and membrane-associated specific receptor proteins. Receptors also may include low molecular weight substances such as dyes or amino acids that are capable of binding to proteins. As a general rule, receptors are the larger of the two components of a binding pair. However, for the purposes of this invention a receptor is to be construed as simply one component of a binding pair.
Ligands are the converse of receptors. While ligands are ordinarily low molecular weight organic compounds they are defined for the purposes of this invention as one component of a binding pair with a receptor in which they may be larger or smaller than the receptor.
Labelled ligand analogues are derivatives of ligands which carry a detectable substituent such as an enzyme, radioisotope or other known label but which are recognizable by and bind to a receptor for the parent ligand with approximately the same affinity as exhibited by the parent ligand.
A ligand-receptor pair is by definition reversibly bindable, i.e., its behavior is governed by the law of mass action. As a practical matter this means that either component of the ligand-receptor pair is displaceable by structurally related substances under the conditions normally encountered in specific binding assays, i.e., mild pH and the temperature and moderate ionic strength. Generally, the ligand-receptor pair will exhibit an affinity constant of greater than 10.sup.7 liters/mole, although useful results may be achieved well outside of this range.
Double receptor, specific binding assays have as their salient feature a first receptor specific for a second receptor, the second receptor ordinarily capable of binding a ligand. This ligand is generally the substance which is being determined in the assay, although it is possible that a third or more receptors may be positioned intermediate to the second receptor and ligand to be assayed. The ligand to be determined is hereinafter designated to be the sample ligand. In the usual context a ligand to be determined is first bound by its receptor, and in turn that receptor is bound by a second receptor. This seeming redundancy has considerable benefit in the analytical arts. The double receptor has been employed in at least three different techniques with the object of improving the separation of sample ligand from solution and simplifying reagent preparation. The first technique is herein termed the universal label method. Many specific binding assays use a labelled receptor in a direct assay, for example the well known sandwich immunoassays. In such methods an excess of insolubilized sample ligand receptor is incubated with a polyepitopic sample until a predetermined proportion of the sample is bound. The insoluble material is then washed and another sample ligand receptor is added. After an additional washing still another receptor is added, this receptor being specific for the second receptor rather than sample ligand. Also, it is labelled with a tag such as a radioisotope, enzyme, stable free radical or other known label so that the extent of its binding to the insoluble complex can be determined. Once again the insoluble material is washed. The amount of labelled second receptor remaining on the insoluble phase or separated in the washing is directly proportional to the amount of sample ligand bound to the ligand receptor and hence is proportional to the concentration of ligand in the sample. Where the last receptor to be added is an antibody, it is conventional to simply use the gamma globulin of a first animal species raised against the gamma globulin of a second, with the latter containing the anti-sample ligand activity. Since this gamma globulin will bind all antibodies from the second species, whatever their specificity for sample ligand, it will serve in labelled form as a universal label, See Beck et al., "Biochem. J." 145:607 (1975). This method has also been previously modified to require only one washing step. As can be seen from the above discussion this technique is directed towards simplifying reagent preparation since only one labelled receptor is needed for the sample ligand receptor, hence all assays can be conducted with a single labelled component.
The two other groups of double receptor, specific binding assays have as their key feature an improvement in the insolubilization of sample ligand receptor, and hence of labelled complexes. One of these assays is disclosed in U.S. Pat. No. 4,092,408. The essence of this method is the use of an insolubilized pair of receptors in place of the previously employed single insoluble receptor in a competitive immunoassay. The method comprises insolubilizing a receptor for sample ligand receptor, sequentially adding sample ligand receptor, then a mixture of labelled sample ligand analogue and sample, followed by separation of the phases and determination of the distribution of labelled sample ligand analogue.
The third group of double receptor specific binding assays are closely related to the preceding method. Instead of supplying an insolubilized receptor for sample ligand receptor during the assay a soluble receptor capable of binding the sample ligand receptor is added to precipitate the same ligand receptor and any sample ligand or labelled sample ligand analogue that may be bound thereto. This technique may be used in a competitive assay, in which case labelled sample ligand analogue is added along with the sample, in a sandwich assay where labelled sample ligand receptor is employed as described above after the sample ligand receptor has been permitted to bind the sample ligand, or in any other conventional system where a sample ligand receptor is a component. Precipitation may be aided by preaggregation of the receptor for sample ligand receptor or by immobilization or absorption of the receptor for sample ligand receptor on an insoluble support. This third group has been traditionally referred to as the double antibody method of phase separation. It is particularly desirable in that it does not affect the amount of labelled analogue which is bound to the sample ligand receptor since the assay is conducted with entirely water soluble reagents up until precipitation occurs.
The double receptor methods have suffered from a number of disadvantages and limitations, principally based on the fact that the affinity of such receptors for one another has been insufficient. This is manifested in requirements for relatively large plastic or solid surfaces for adsorption of receptor, making it particularly difficult to use in a coated tube, and for extensive and prolonged incubations, thus greatly extending the overall time required for completion of an assay. These low affinities are particularly critical where receptor or the antigen used to raise the sample ligand receptor is difficult to obtain.
These disadvantages have largely precluded the use of insoluble double receptors in automated rechargeable radioimmunoassay systems. These systems are characterized by the cyclical recharge of a conventional insoluble receptor after each assay by eluting the receptor-bound substances, e.g., sample ligand and labelled ligand analogue. These systems are handicapped particularly where large molecules such as antigens are to be determined. These molecules are less likely to penetrate to receptor sites hidden in a carpet of receptor spread across the insoluble surface. This results in low sample ligand receptor affinity and in turn a requirement for larger absorption area or longer reaction times. This clearly presents a severe problem when speed and sensitivity are paramount.
Furthermore, such rechargeable systems are rarely completely rechargeable, i.e., it is difficult to achieve complete elution of sample ligand or labeled sample ligand analogue, particularly where the receptor binding site is amphoteric. Further, while eluting reagents containing salts and extreme pH may remove ligands they will also gradually irreversibly denature the receptors, thereby making them unfit for reuse.
Accordingly it is an object of this invention to supply a readily rechargeable receptor for automated specific binding assays.
It is an additional object to provide a labelled universal receptor having improved affinity.
It is a further object to improve the affinity of double receptor, specific binding assays for sample ligands.
It is also an object to accelerate phase separations in double receptor assays.
It is another object to reduce the surface area needed for double receptor assays to bind sufficient sample ligand.
Other objects of this invention will be apparent to those skilled in the art from a consideration of this specification taken in its entirety.